27-Hydroxycholesterol regulates human SLC22A12 gene expression through estrogen receptor action.

The excretion and reabsorption of uric acid both to and from urine are tightly regulated by uric acid transporters. Metabolic syndrome conditions, such as obesity, hypercholesterolemia, and insulin resistance, are believed to regulate the expression of uric acid transporters and decrease the excretion of uric acid. However, the mechanisms driving cholesterol impacts on uric acid transporters have been unknown. Here, we show that cholesterol metabolite 27-hydroxycholesterol (27HC) upregulates the uric acid reabsorption transporter URAT1 encoded by SLC22A12 via estrogen receptors (ER). Transcriptional motif analysis showed that the SLC22A12 gene promoter has more estrogen response elements (EREs) than other uric acid reabsorption transporters such as SLC22A11 and SLC22A13, and 27HC-activated SLC22A12 gene promoter via ER through EREs. Furthermore, 27HC increased SLC22A12 gene expression in human kidney organoids. Our results suggest that in hypercholesterolemic conditions, elevated levels of 27HC derived from cholesterol induce URAT1/SLC22A12 expression to increase uric acid reabsorption, and thereby, could increase serum uric acid levels.

Inhibition of the ATR kinase enhances 5-FU sensitivity independently of nonhomologous end-joining and homologous recombination repair pathways.

The anticancer agent 5-fluorouracil (5-FU) is cytotoxic and often used to treat various cancers. 5-FU is thought to inhibit the enzyme thymidylate synthase, which plays a role in nucleotide synthesis and has been found to induce single- and double-strand DNA breaks. ATR Ser/Thr kinase (ATR) is a principal kinase in the DNA damage response and is activated in response to UV- and chemotherapeutic drug-induced DNA replication stress, but its role in cellular responses to 5-FU is unclear. In this study, we examined the effect of ATR inhibition on 5-FU sensitivity of mammalian cells. Using immunoblotting, we found that 5-FU treatment dose-dependently induced the phosphorylation of ATR at the autophosphorylation site Thr-1989 and thereby activated its kinase. Administration of 5-FU with a specific ATR inhibitor remarkably decreased cell survival, compared with 5-FU treatment combined with other major DNA repair kinase inhibitors. Of note, the ATR inhibition enhanced induction of DNA double-strand breaks and apoptosis in 5-FU-treated cells. Using gene expression analysis, we found that 5-FU induced the activation of the intra-S cell-cycle checkpoint. Cells lacking BRCA2 were sensitive to 5-FU in the presence of ATR inhibitor. Moreover, ATR inhibition enhanced the efficacy of the 5-FU treatment, independently of the nonhomologous end-joining and homologous recombination repair pathways. These findings suggest that ATR could be a potential therapeutic target in 5-FU-based chemotherapy.

Identification of three distinct cell populations for urate excretion in human kidneys.

In humans, uric acid is an end-product of purine metabolism. Urate excretion from the human kidney is tightly regulated by reabsorption and secretion. At least eleven genes have been identified as human renal urate transporters. However, it remains unclear whether all renal tubular cells express the same set of urate transporters. Here, we show renal tubular cells are divided into three distinct cell populations for urate handling. Analysis of healthy human kidneys at single-cell resolution revealed that not all tubular cells expressed the same set of urate transporters. Only 32% of tubular cells were related to both reabsorption and secretion, while the remaining tubular cells were related to either reabsorption or secretion at 5% and 63%, respectively. These results provide physiological insight into the molecular function of the transporters and renal urate handling on single-cell units. Our findings suggest that three different cell populations cooperate to regulate urate excretion from the human kidney, and our proposed framework is a step forward in broadening the view from the molecular to the cellular level of transport capacity.

Successful treatment of streptococcal toxic shock syndrome complicated by primary peritonitis and bilateral empyema in a healthy young woman: Identification of uncommon clone emm103 and novel sequence type 1363.

Streptococcal toxic shock syndrome (STSS) has a dramatic clinical course and high mortality rate. Here, we report a case of STSS complicated by primary peritonitis and bilateral empyema. A previously healthy young woman was diagnosed with STSS complicated by primary peritonitis and bilateral empyema. Blood culture results on admission were negative. Sever shock, respiratory failure, systemic inflammation, thrombocytopenia, renal failure, ascites, and pleural effusion occurred, mimicking thrombocytopenia, anasarca, fever, reticulin fibrosis/renal failure and organomegaly (TAFRO) syndrome. Retesting blood cultures identified Streptococcus pyogenes. Gram staining of ascites and pleural fluid indicated gram-positive cocci in chains. Antibiotics, immunoglobulins, and surgical intervention led to recovery without complications. Ex-post genotypic analyses showed uncommon emm103.0 (cluster E3) of emm long sequence (784 base) and novel sequence type 1363. STSS diagnosis can be difficult as it mimics other systemic inflammatory diseases. Therefore, it is crucial for clinicians to perform microbiological examinations from infection foci, even if the initial culture is negative.

Gene Expression Profiles of Human Cerebral Organoids Identify PPAR Pathway and PKM2 as Key Markers for Oxygen-Glucose Deprivation and Reoxygenation.

Ischemic stroke is one of the most common neurological diseases. However, the impact of ischemic stroke on human cerebral tissue remains largely unknown due to a lack of ischemic human brain samples. In this study, we applied cerebral organoids derived from human induced pluripotent stem cells to evaluate the effect of oxygen-glucose deprivation/reoxygenation (OGD/R). Pathway analysis showed the relationships between vitamin digestion and absorption, fat digestion and absorption, peroxisome proliferator-activated receptor (PPAR) signaling pathway, and complement and coagulation cascades. Combinational verification with transcriptome and gene expression analysis of different cell types revealed fatty acids-related PPAR signaling pathway and pyruvate kinase isoform M2 (PKM2) as key markers of neuronal cells in response to OGD/R. These findings suggest that, although there remain some limitations to be improved, our ischemic stroke model using human cerebral organoids would be a potentially useful tool when combined with other conventional two-dimensional (2D) mono-culture systems.

Brainstem Organoids From Human Pluripotent Stem Cells.

The brainstem is a posterior region of the brain, composed of three parts, midbrain, pons, and medulla oblongata. It is critical in controlling heartbeat, blood pressure, and respiration, all of which are life-sustaining functions, and therefore, damages to or disorders of the brainstem can be lethal. Brain organoids derived from human pluripotent stem cells (hPSCs) recapitulate the course of human brain development and are expected to be useful for medical research on central nervous system disorders. However, existing organoid models are limited in the extent hPSCs recapitulate human brain development and hence are not able to fully elucidate the diseases affecting various components of the brain such as brainstem. Here, we developed a method to generate human brainstem organoids (hBSOs), containing midbrain/hindbrain progenitors, noradrenergic and cholinergic neurons, dopaminergic neurons, and neural crest lineage cells. Single-cell RNA sequence (scRNA-seq) analysis, together with evidence from proteomics and electrophysiology, revealed that the cellular population in these organoids was similar to that of the human brainstem, which raises the possibility of making use of hBSOs in investigating central nervous system disorders affecting brainstem and in efficient drug screenings.

Six-month cultured cerebral organoids from human ES cells contain matured neural cells.

Recently, researchers have developed protocols for human cerebral organoids using human pluripotent stem cells, which mimic the structure of the developing human brain. Existing research demonstrated that human cerebral organoids which undergo short cultivation periods, contain astrocytes, neurons, and neural stem cells, but lacked mature oligodendrocytes, and mature, fully functional neurons. In this study, we analyzed organoids induced from H9 human embryonic stem (ES) cells that were cultivated for as long as six months. We observed mature oligodendrocytes, positive for MBP (myelin-basic protein), and mature GAD67 (glutamate decarboxylase 67 kDa isoform)-positive inhibitory neurons and VGLUT1 (vesicular glutamate transporter 1)-positive excitatory neurons via immunohistochemical analysis. These observations suggest that long-term cultivation of cerebral organoids can lead to the maturation of human cerebral organoids, which can be used as a tool to study the development of human brains.